Physicochemical Characterization of a Biomimetic, Elastin-Inspired Polypeptide with Enhanced Thermoresponsive Properties and Improved Cell Adhesion

Genetic engineering allows fine-tuning and controlling protein properties, thus exploiting the new derivatives to obtain novel materials and systems with improved capacity to actively interact with biological systems. The elastin-like polypeptides are tunable recombinant biopolymers that have proven to be ideal candidates for realizing bioactive interfaces that can interact with biological systems. They are characterized by a thermoresponsive behavior that is strictly related to their peculiar amino acid sequence. We describe here the rational design of a new biopolymer inspired by elastin and the comparison of its physicochemical properties with those of another already characterized member of the same protein class. To assess the cytocompatibility, the behavior of cells of different origins toward these components was evaluated. Our study shows that the biomimetic strategy adopted to design new elastin-based recombinant polypeptides represents a versatile and valuable tool for the development of protein-based materials with improved properties and advanced functionality.


FIGURE 1S
Primary structure of the three human elastin-like polypeptides described in this paper.The monomers (in brackets) are repeated eight times in the final construct.In black, the his-tag at the N-terminus is evidenced.Boxed, the pentapeptidic (orange) and the hexapeptidic (red) repeats.

FIGURE 2S
Size distribution of the apparent hydrodynamic diameter (Dh) determined as scattering intensity (red line) and volume (blue line) at 15 °C for the following 2 mg/ml biopolymer solutions:

D FIGURE 3S
Chemical shift of resonance peaks as a function of temperature for the different amino acidic residues of HELP (A) and UELP (B)

FIGURE 4S
Crystal violet adhesion assays of MG 63 (upper panel) and NIH3T3 (lower panel) cells at 24 hours after seeding.Non-adhesive tissue culture polystyrene wells were left uncoated (control) or were coated HELP and UELP, respectively, like in Figure 10 and Figure 11.The control was the culture on tissue culture treated polystyrene.* * * p < 0.0001; n = 5 via one-way ANOVA.
Representative phase contrast images of MG63 osteoblastic cells of human origin cultured on non treated polystyrene (NP) surfaces coated by incubation with aqueous solutions with different concentration of UELP biopolymer (on the left, boxed in black).Control cultures on tissue culture treated polystyrene (TP, green box), on NP without any biopolymer (blue box) as well as cultures on NP coated with HELP (red box) were carried on in parallel (on the top).
The coatings were prepared in a non-treated 96-well microplate by adding 100 µl of 0.22 µ -filtered aqueous solutions of biopolymer at 0.0004, 0.004, 0.04, and 0.4% (w/v) per well.After overnight incubation at 5°C, the solution was removed, and the wells were washed twice with 200 µL of sterile water and then air-dried.100 µL of supplemented DMEM containing 5000 cells were seeded in each well.After 24 hours, the cultures were inspected by phase contrast microscopy and the images were acquired.
Cell viability after 24 hours was assessed by the WST-1 metabolic assay.Depending on the coating, after 24 hours two cells morphologies were observed in the cultures: unattached, rounded cells and flat and spread cells, attached to the surface.Thus, the assay was performed under two conditions, one to assess the viability of the culture (attached and unattached cells) and the other to assess the viability of the attached cells only.
A -Culture viability: 24 hours after seeding, 5 µL of WST-1 reagent were directly added to the 100 µL of culture medium in each well.After 90 min of incubation at 37°C, the absorbance was measured at 450 nm by a microplate reader.
B -Viability of the attached cells: 24 hours after seeding, the culture medium was removed and cell were washed with 100 µl of sterile PBS to remove all non-attached cells.Then, 100 µl of supplemented DMEM containing 5 µl of WST-1 reagent were added per well.After 90 min of incubation at 37°C, the absorbance was measured at 450 nm by a microplate reader.
Values were normalized to the TP control cultures.* * * p < 0.0001; n = 8 via one-way ANOVA.
Representative phase contrast images of NIH3T3 cells cultured on non treated polystyrene (NP) surfaces coated by incubation with aqueous solutions with different concentration of UELP biopolymer (on the left, boxed in black).Control cultures on tissue culture treated polystyrene (TP, green box), on NP without any biopolymer (blue box) as well as cultures on NP coated with HELP (red box) were carried on in parallel (on the top).
The same conditions and procedures already described for MG63 cell culture in the previous Figure 5S were employed for NIH3T3 murine fibroblasts.
A -Culture viability B -Viability of the attached cells Values were normalized to the TP control cultures.* * * p < 0.0001; n = 8 via one-way ANOVA.